Cortico-Accumbens Regulation of Approach-Avoidance Behavior Is Modified by Experience and Chronic Pain  Neil Schwartz, Catriona Miller, Howard L. Fields  Cell Reports  Volume 19, Issue 8, Pages 1522-1531 (May 2017) DOI: 10.1016/j.celrep.2017.04.073 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 19, 1522-1531DOI: (10.1016/j.celrep.2017.04.073) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 Experience and Chronic Pain Regulate Avoidance Elicited by a Pain-Predictive Cue during Reward-Directed Behavior (A) Schematic of a rat in the testing chamber in which an infra-red laser can apply a benign (blue) or noxious (red) heat stimulus to the animal’s fore-shoulder during consumption of either a small (3%) or large (10%) sucrose reward. (B) Task sequence: tone 1 indicates that sucrose is available, a lick in response to tone 1 initiates a trial and delivery of a smaller or larger reward; 1.5 s after the first lick and on 85% of the trials the PPC is played; 1 s after the onset of the PPC, there is a 30% probability of a noxious heat stimulus. A detailed timeline is in Figure S1. (C) Timeline of the four testing sessions. During pre-training, all auditory cues were presented with benign fore-shoulder stimulation. Next, as described in (B), the noxious heat stimulus was introduced. Training on the task with the noxious stimulus continued for 6–9 days. Next, animals underwent either SNI or sham surgeries, and 7 days later testing resumed. (D and E) Time course (D) and average (E) of normalized lick rates for trials with the small reward during each of the four testing session described in (C). Symbols are means, vertical lines indicate SEM, the horizontal dashed line indicates the baseline lick rate, and the solid line indicates the epoch used for analysis in (E). The blue, orange, and black lines at the bottom of each graph indicate sucrose, PPC, and benign fore-shoulder stimulation, respectively. Note that the avoidance score is the additive inverse of the normalized lick rate. (F and G) Plots as in (D) and (E) for large reward trails. Changes in avoidance between the four sessions show that training reduces avoidance during the PPC, and in the SNI model PPC-avoidance is selectively reinstated during consumption of the small reward (pre-training, n = 11; early, n = 22; trained, n = 22, SNI, n = 7). ∗∗∗p < 0.001, ###p < 0.001, Bonferroni post hoc t test. All error bars are SEM. Cell Reports 2017 19, 1522-1531DOI: (10.1016/j.celrep.2017.04.073) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 The IL Cortex Is Required for Suppression of PPC-Avoidance during Reward-Directed Behavior (A) Timeline of cortical inactivation experiments. (B) Injection sites of GABA agonists or areas of highest expression of the inhibitory DREADDs channel hM4Di. (C) In trained animals, inactivation of the IL-cortex (but not prelimbic or anterior cingulate) reinstated PPC-avoidance during consumption of both rewards (rats: PrL: n = 4 [3 Baclofen and Muscimol; B&M, 1 DREADDS]; ACC: n = 5 [2 B&M, 3 DREADDS]; IL: n = 7 [3 B&M, 4 DREADDS]). ∗p < 0.05, ∗∗p < 0.001, Bonferroni post hoc t test. Controls, blinding, and counter balancing of the injections are described in the Supplemental Experimental Procedures. Cell Reports 2017 19, 1522-1531DOI: (10.1016/j.celrep.2017.04.073) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 Spiking in Two Cell Types in the IL-Cortex Predicts Suppression of PPC-Avoidance during Reward-Directed Behavior (A) Proportion of PPC-responsive IL pyramidal neurons. Rats: early and trained, n = 7; SNI, n = 5. (B and C) Heatmap of spiking: top left and bottom right panels are neurons spiking during the PPC and consumption of the small reward and large reward, respectively. Adjacent plots show that these neurons exhibit little spiking during the PPC and consumption of the alternate reward, with summary data in (C). The dashed vertical line indicates the start of the PPC. Note that only 6 of 82 neurons (7.3%) increase spiking during the PPC and consumption of both rewards, implying that there are two distinct subpopulations of PPC-responsive neurons. (D) Spike rates and corresponding PPC-avoidance score for trials in (B) with the linear regression lines for PPC + small and PPC + large reward responsive neurons: y = −0.54 + 0.17, R2 = 0.6 and y = −0.56 + 0.2, R2 = 0.9, respectively. Vertical and horizontal lines are SEM. PPC-evoked spiking in both subpopulations is positively correlated with suppressed PPC-avoidance (i.e., maintained consumption of the rewards). (E and F) Timeline of three recording sessions, with summary data in (F) showing that once trained, mean spike rates increase. However, in the SNI model, there is a selective decrease in PPC + small reward responsive neurons. ∗p < 0.05, #p < 0.05; ∗∗p < 0.01, Bonferroni post hoc t test. The time course of PPC-evoked spiking, the individual data points for (D), and placements are in Figure S4. Cell Reports 2017 19, 1522-1531DOI: (10.1016/j.celrep.2017.04.073) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 Spiking in Two Cell Types in the NAc Predicts Suppression of PPC-Avoidance during Reward-Directed Behavior (A) Proportion of PPC-responsive MSNs in the NAc. Rats: early and trained, n = 7; SNI, n = 5. (B and C) Heatmaps of spiking during consumption of the small reward (left) and large reward (right), with summary data in (C). The dashed vertical line indicates the start of the PPC. Note that only 16 of 136 PPC-responsive neurons (11.8%) increased spiking during the PPC and consumption of both reward, implying that there are two subpopulations of PPC-responsive neurons. (D) Linear regression of spike rates and PPC-avoidance score for trials in (B) show that much like spiking in the IL-cortex, PPC evoked spiking in two subpopulations of neurons in the NAc predicts that the animal will suppress PPC-avoidance during reward-directed behavior (i.e., maintain lick speed). PPC + small and PPC + large reward: y = −0.18 + 0.15, R2 = 0.7 and y = −0.08 + 0.15, R2 = 0.6, respectively. Vertical and horizontal bars indicate SEM. (E) Timeline of three recording sessions. (F) Summary spiking data for each session (early, trained, and SNI), showing that in the SNI model, spiking of PPC + small reward-responsive MSNs is reduced compared to large reward-responsive MSNs. #p < 0.05, Bonferroni post hoc t test. The time course of PPC-evoked spiking, the individual data points for (D), and placements are in Figure S5. Cell Reports 2017 19, 1522-1531DOI: (10.1016/j.celrep.2017.04.073) Copyright © 2017 The Author(s) Terms and Conditions

Figure 5 IL-NAc Terminals Regulate PPC-Avoidance during Reward-Directed Behavior (A) Example of DREADDs (hM4Di) expression in the IL-cortex and timeline of chemogenetic inactivation while recording PPC-responsive MSNs in the NAc of trained animals. cc, corpus callosum; PrL, pre-limbic; v, olfactory ventricle. (B) Proportion of PPC-responsive MSNs; electrode positions in the NAc are in Figure S5. (C and D) Time course of PPC-evoked spiking (C) shows that IL-inactivation reduces PPC-evoked spiking in both PPC-small and PPC-large reward-responsive MSNs in the NAc (n = 7 and 6, respectively as indicated by the vertical line to the left of the heat plot). Insets above average time course indicate the period used in (D) to compare average MSN spiking and PPC-avoidance. Silencing the IL reduced PPC-evoked spiking in MSNs (n = 13 MSNs from two rats). ∗∗p < 0.01, paired t test. Note that behavioral data for n = 7 rats is in Figure 2. (E) Timeline and example Chr2 expression in the IL for recording MSN responses to opto-stimulation of IL axons in the NAc before the task in trained animals. (F) Proportion of MSNs recorded during both opto-stimulation and during the task (n = 2 trained rats on days 8 and 9; four sessions). (G and H) Time course and summary data demonstrate that intra-NAc activation of IL-axons terminals increases spiking of PPC-responsive MSNs. ∗p = 0.04, t test. Heatmaps corresponding to (G) are in Figure S6. Cell Reports 2017 19, 1522-1531DOI: (10.1016/j.celrep.2017.04.073) Copyright © 2017 The Author(s) Terms and Conditions

Figure 6 IL-NAc Terminals Regulate PPC-Avoidance during Goal-Directed Behavior (A) Timeline, example of bilateral Chr2 expression in the IL, and optical fiber positions in the NAc for bilateral opto-stimulation of IL-NAc axon terminals after induction of the SNI model, a time point when PPC-avoidance is reinstated. cc, corpus callosum; PrL, pre-limbic; v, olfactory ventricle. (B) Schematic of interleaved Chr2 stimulation trials post-induction of the SNI model. In the same session, IL-axons in the NAc were stimulated either before or during the PPC. (C and D) Time course showing that optical stimulation does not affect baseline licking but increases licking during the PPC; i.e., as shown in (D) optical stimulation of IL terminals in the NAc during the PPC, reduces PPC-avoidance post-induction of the SNI model (n = 4 rats) ∗p = 0.03, paired t test. These results indicate that IL-NAc terminal activity is sufficient to suppress PPC-avoidance. Cell Reports 2017 19, 1522-1531DOI: (10.1016/j.celrep.2017.04.073) Copyright © 2017 The Author(s) Terms and Conditions